Sterol derivatives, liposomes comprising sterol derivatives and method of loading liposomes with active substances

ABSTRACT

A sterol derivative with a pKa value of between 3.5 and 8, according to the general formula cation-spacer 2-Y-spacer 1-X-sterol, wherein Y and X represent linking groups, is suggested, as well as liposomes comprising such sterol derivatives.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.10/468,652, filed Feb. 11, 2004, which is a §371 national stage ofPCT/EP02/01879, filed Feb. 21, 2002, which claims priority to GermanPatent Application Number 101 09 898.7 filed Feb. 21, 2001, all of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to polar compounds based on a sterol skeleton, the3-position of the ring system being substituted by an organic cationhaving a pK value of between 3.5 and 8. The invention also relates toliposomes containing such compounds.

BACKGROUND OF THE INVENTION

The term “lipids” summarizes three classes of natural materials whichcan be isolated from biological membranes: phospholipids, sphingolipids,and cholesterol, including its derivatives.

These substances are of technical interest in the production ofliposomes. Inter alia, such liposomes can be used as containers foractive substances in pharmaceutical preparations. In such uses,efficient and stable packaging of the cargo and controllable release ofthe contents are desirable. Both of these requirements are not easy tocombine: the more stable and compact the packaging, the more difficultthe release of the entrapped active substance therefrom. For thisreason, liposomes changing their properties in response to an externalstimulus have been developed. Thermosensitive and pH-sensitive liposomesare well-known. The pH-sensitive liposomes are of special interest,because this parameter undergoes changes even under physiologicalconditions, e.g. during endocytotic reception of a liposome in a cell,or during passage of the gastrointestinal tract.

The following abbreviations will be used hereinafter:

CHEMS Cholesterol hemisuccinate

PC Phosphatidyl choline

PE Phosphatidyl ethanolamine

PS Phosphatidyl serine

His-Chol Histaminylethaneamine-cholesterol hemisuccinate

Py-Chol Pyridylethaneamine-cholesterol hemisuccinate

Mo-Chol Morpholinoethaneamine-cholesterol hemisuccinate

PDEA-Chol Pyridyldithioethaneamino-cholesterol hemisuccinate

According to the prior art, pH-sensitive liposomes particularly compriseCHEMS. CHEMS, in mixture with phosphatidyl ethanolamine, is used toproduce pH-sensitive liposomes (Tachibana et al. (1998); BBRC 251,538-544, U.S. Pat. No. 4,891,208). Such liposomes can enter cells byendocytosis and are capable of transporting cargo molecules into theinterior of cells on this route, without doing damage to the integrityof the cellular membrane.

One substantial drawback of CHEMS is its anionic character. Liposomesproduced using same have a negative overall charge and,disadvantageously, are taken up by cells with low efficiency. Despitethe transfer mechanism described above, they are barely suitable for thetransport of macromolecules into cells.

For this purpose, the art uses cationic liposomes having a preferablyhigh and constant surface charge. The positive overall charge of suchparticles leads to electrostatic adherence to cells and subsequently toefficient transport into same. The use of these compounds and ofliposomes produced using same remains restricted to in vitro or ex vivoapplications, because such positively charged liposomesdisadvantageously result in uncontrolled formation of aggregates withserum components.

SUMMARY OF THE INVENTION

The object was therefore to produce new compounds,

i) by means of which active substances can be entrapped in liposomes andreleased therefrom when changing the pH value; and

ii) the presence of which aids to achieve the production of cationicliposomes which can be mixed with serum without formation of largeraggregates.

Other objects of the invention involve finding ways allowing easy andlow-cost production of the desired compounds and incorporation thereofin high amounts in liposomal membranes.

The object of the invention is accomplished by means of a sterolderivative with a pKa value of between 3.5 and 8, according to thegeneral formula:

Cation-Spacer 2-Y-Spacer 1-X-Sterol,

wherein Y and X represent linking groups. Depending on the cation used,compounds are obtained which undergo changes in their charge at aspecific pH value owing to the sterol component and allow incorporationthereof in liposomal membranes in high amounts. Ordinary and inexpensivesterols or derivatives thereof can be used as starting compounds.Accordingly, the object of the invention can be accomplished byconjugating pH-sensitive cations to the 3-position of a sterol skeleton.

Among the membrane-forming or membrane-bound groups of a biologicalbilayer membrane, the sterols are of special interest because thesecompounds are available at low cost, involve ordinary chemistry, andallow incorporation in membranes in high amounts without increasing thepermeability thereof or even completely destroying their membranecharacter. However, in order to retain this latter feature, it isimportant that substitution with a polar molecule be at the 3-positionof the sterol.

The cation or cationic group can be e.g. a nitrogen base. The sterol ischolesterol, for example. Situated between the cationic group and thesterol skeleton are the molecule fragments spacer 2-Y-spacer 1-X.

For example, spacer 1 is a lower alkyl residue of linear structure,which has 8 C atoms and includes e.g. 2 ethylenically unsaturated bonds.Spacer 2 is e.g. a lower alkyl residue of linear structure, which mayhave 8 C atoms and includes 2 ethylenically unsaturated bonds.

The overall molecule assumes its pH-dependent charge characteristics byone or more organic cations with a pKa value between 3.5 and 8. Typicalmolecules or molecule fragments with this property are nitrogen bases.These nitrogen bases are linked to the 3-position of the sterol skeletonvia spacers and coupling groups, thus forming a compound according tothe formula of the invention. In many cases, e.g. where the nitrogenbases are in the form of a ring system, positional isomers are existing,wherein the linking spacer is substituted to various positions of theorganic cation. Such positional isomers fall within the disclosure ofthis invention. In many cases, the pKa values of the organic cation canbe influenced via said positional isomerism alone. The relevantfundamental rules are well-known to those skilled in the art.Alternatively, these effects can be estimated from tabular compilations(Handbook of Chemistry and Physics, Vol. 73, pp. 8-37ff.).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A. Structural formula of Histidineamido-cholesterolhemisuccinate, m.w. 580 g/mol; B. structural formula ofPyridyldithioethaneamido-cholesterol hemisuccinate, m.w. 655 g/mol.

FIG. 2: Shows transfection of HeLa cells according to Example 12: A.Transfection by TRITC dextran with liposomes (DOPE 60/His-Chol 40); B.Transfection by TRITC dextran with liposomes (POPC 60/His-Chol 40).

DETAILED DESCRIPTION

In a preferred embodiment of the invention, the sterol derivative has apKa value of between 4 and 6.5. Advantageously, this pKa value falls ina range which is of crucial importance for the physiology of numerousorganisms.

In another preferred embodiment of the invention, the cations arenitrogen bases. The cations preferably can be derived from piperazines,imidazoles, morpholines, purines and/or pyrimidines.

Coupling reactions result in amphiphilic organic cations, e.g. thosederived from the following classes of substances:

o-, m-, p-anilines; 2-, 3- or 4-substituted anisidines, toluidines orphenetidines; 2-, 3-, 5-, 6-, 7- or 8-substituted benzimidazoles, 2-,3-, 4- or 5-substituted imidazoles, 1- or 5-substituted isoquinolines,2-, 3- or 4-substituted morpholines, 2-, 3- or 4-substituted picolines,1-, 2- or 3-substituted piperazines, 2-, 5- or 6-modified pterines, 3-,4-, 5-, 6- or 9-substituted purines, 2- or 3-substituted pyrazines, 3-or 4-substituted pyridazines, 2-, 3- or 4-modified pyridines, 2-, 4-, 5-or 6-substituted pyrimidines, 1-, 2-, 3-, 4-, 5-, 6- or 8-substitutedquinolines, 2-, 4- or 5-substituted thiazoles, 2-, 4- or 6-substitutedtriazines, or derivatives of tyrosine. Particularly preferred arepiperazines, imidazoles, morpholines, purines and/or pyrimidines.

Highly preferred are molecule fragments such as occurring in biologicalsystems, i.e., for example: 4-imidazoles (histamines), 2-, 6- or9-purines (adenines, guanines, adenosines, or guanosines), 1-, 2- or4-pyrimidines (uracils, thymines, cytosines, uridines, thymidines,cytidines), or pyridine-3-carboxylic acids (nicotinic esters or amides).

The above-mentioned structural fragments may also have additionalsubstituents. For example, these can be methyl, ethyl, propyl, orisopropyl residues, more preferably in hydroxylated form, including oneor two hydroxyl groups. Also, these can be hydroxyl or keto functions inthe ring system. In addition, other structural fragments are alsopossible unless anionically dissociated molecule portions are formed ina pH range between 3.5 and 8.5, e.g. carboxylic acids, sulfonic acids,or some aromatic hydroxyl groups or enols.

Nitrogen bases with preferred pKa values are also formed by single ormultiple substitution of the nitrogen atom with lower alkanehydroxylssuch as hydroxymethyl or hydroxyethyl groups. Suitable organic basesfrom this group are e.g. aminopropanediols, triethanolamines,tris(hydroxymethyl)methylamines, bis(hydroxymethyl)methylamines,tris(hydroxyethyl)methylamines, bis(hydroxyethyl)methylamines, or thecorresponding substituted ethylamines. Coupling of these fragments tothe hydrophobic portion of the molecule may proceed either via thenitrogen of the base or via any of the hydroxyl functions.

In addition to sterol derivatives including a single organic cation,those including two or three identical or different groups are alsopreferred. All of these groups are required to have a pKa value in theabove-mentioned range. One suitable complex group is the amide ofhistamine and histidine or of histamine and histidylhistidine.

Anionic groups such as carboxylic acids, sulfonic acids, enols, oraromatic hydroxyls are allowable as component of the molecule only ifundissociated in the claimed pH range between 3.5 and 8.5. In general,this is the case if the pKa value is above 9.5.

In another preferred embodiment of the invention, the linking group Xhas the structure —(C═O)—O—; —(C═O)—NH—; —(C═O)—S—; —O—; —NH—; —S—; or—CH═N—, for example. In particular, the linking group Y corresponds inits structure to the group X, and may additionally assume the structure—O—(O═C)—; —S—(O═C)—; —NH—(O═C)—; or —N═CH—. The Y group can be omittedin those cases where the organic cation can be coupled directly to thesterol skeleton, e.g. in the esterification of 4-imidazoleacetic acidwith cholesterol.

In another preferred embodiment of the invention, spacer 1 is a loweralkyl residue of linear, branched or cyclic structure, which has from 1to 8 C atoms and includes 0, 1 or 2 ethylenically unsaturated bonds.Spacer 1 may have hydroxyl groups so as to increase the polarity of themolecule. In particular, spacer 1 can be a sugar. Spacer 2 is a loweralkyl residue of linear, branched or cyclic structure, which has from 0to 8 C atoms and includes 0, 1 or 2 ethylenically unsaturated bonds.Spacer 2 may have hydroxyl groups so as to increase the polarity of themolecule. In particular, spacer 2 can be a sugar.

Methods of performing such coupling reactions are well-known to thoseskilled in the art and may vary depending on the starting material andcoupling component employed. Typical reactions are esterification,amidation, addition of amines to double bonds, etherification, orreductive amination.

A particularly preferred method of coupling is amidation of sterolhemisuccinates. Inter alia, molecules satisfying the requirementsaccording to the object of the invention can be produced by coupling ofhistamine, N-(2-aminoethyl)morpholine, N-(2-aminoethyl)piperazine,N-(2-aminoethyl)pyridine, or pyridyldithioethenylamine. The compoundsthus obtained will be referred to as His-Chol, Mo-Chol, Pip-Chol,Py-Chol, or PDEA-Chol herein.

In another preferred embodiment of the invention, the sterols areparticularly cholesterol, sitosterol, campesterol, desmosterol,fucosterol, 22-ketosterol, 20-hydroxysterol, stigmasterol,22-hydroxycholesterol, 25-hydroxycholesterol, lanosterol,7-dehydrocholesterol, dihydrocholesterol, 19-hydroxycholesterol,5α-cholest-7-en-3β-ol, 7-hydroxycholesterol, epicholesterol, ergosterol,and/or dehydroergosterol, as well as other related compounds.

The sterols that are used may bear various groups in the 3-positionthereof, which groups allow for ready and stable coupling or optionallyassume the function of a spacer. Particularly suitable for directcoupling are the hydroxyl group which is naturally present, but also,the chlorine of sterol chlorides, or e.g. the amino group ofsterolamines, or the thiol group of thiocholesterol.

The invention also relates to liposomes comprising the substancesaccording to the invention. All of the substances or compounds of theinvention can be incorporated in high amounts in liposomal membranes,resulting in a positive charge of the overall particle only if the pHvalue of the medium is smaller than (pKa+1) of the compounds accordingto the invention.

In a special embodiment of the invention, the amount of sterolderivative is 50 mole-% at maximum. Compositions including at least 5mole-% of compound, but 40 mole-% at maximum, are particularlypreferred. Compositions including at least 10 mole-% of sterolderivative and 30 mole-% at maximum are highly preferred.

Another embodiment wherein the liposomes specifically comprisephosphatidyl choline, phosphatidyl ethanolamine and/or diacylglycerol isalso convenient. Cholesterols themselves are incapable of formingliposomes, and therefore, addition of further lipid is necessary. Inparticular, this lipid can be a phospholipid. Obviously, furthermodifications of the liposome are possible. Thus, the use ofpolyethylene glycol-modified phospholipids or analogous products isparticularly advantageous.

In another embodiment of the invention, the liposomes have an averagesize of between 50 and 1000 nm, preferably between 50 and 300 nm, andmore preferably between 60 and 130 nm.

In another preferred embodiment, the liposomes comprise activesubstances. For example, the liposomes according to the invention aresuitable for parenteral application. They can be used e.g. in cancertherapy and in the therapy of severe infections. To this end, liposomedispersions can be injected, infused or implanted. Thereafter, they aredistributed in the blood or lymph or release their active substance in acontrolled fashion as a depot. The latter can be achieved by highlyconcentrated dispersions in the form of gels. The liposomes can also beused for topical application on the skin. In particular, they maycontribute to improved penetration of various active substances into theskin or even passage through the skin and into the body. Furthermore,the liposomes can also be used in gene transfer. Due to its size andcharge, genetic material is usually incapable of entering cells withoutan aid. For this purpose, suitable carriers such as liposomes or lipidcomplexes are required which, together with the DNA, are to be taken upby the respective cells in an efficient and well-directed fashion. Tothis end, cell-inherent transport mechanisms such as endocytosis areused. Obviously, the liposomes of the invention can also be used asmodel membranes. In their principal structure, liposomes are highlysimilar to cell membranes. Therefore, they can be used as membranemodels to quantify the permeation rate of active substances throughmembranes or the membrane binding of active substances.

Advantageously, liposomes produced using the substances of the inventionshow low non-specific binding to cell surfaces. It is this lownon-specific binding which is an essential pre-condition for achievingspecific binding to target cells. Target control of the vehicles isobtained when providing the above-described liposomes with additionalligands. As a result, the active substance can be accumulatedspecifically in such cells or tissues which exhibit a pathologicalcondition.

One important use of the substances according to the invention istherefore in the construction of vectors for transfer of activesubstances in living organisms. The vectors are particularly suited forthe transport of therapeutic macromolecules such as proteins or DNAwhich themselves are incapable of penetrating the cell membrane orundergo rapid degradation in the bloodstream.

Advantageously, antibodies, lectins, hormones or other active substancescan be coupled to the surface of liposomes under mild conditions in highyields. In one variant of the teaching according to the invention, theliposomes for such a use comprise a sufficient amount of PDEA-Chol inaddition to other lipids, including those mentioned in the presentspecification. The amount of PDEA-Chol employed will depend on thedesired use. Thus, liposomes loaded with a marker require a high ratioof this signal generator to the component determining the specificity.In this case, only a low number of antibodies per liposome have to becoupled.

In a preferred embodiment of the invention, the liposomes comprise aprotein, a peptide, a DNA, an RNA, an antisense nucleotide, and/or adecoy nucleotide as active substance.

In a particularly preferred embodiment of the invention, at least 80% ofthe active substance is inside the liposome.

The invention also relates to a method of loading liposomes with activesubstances, wherein one defined pH value is used for encapsulation, anda second pH value is adjusted to remove unbound active substance.

The invention also relates to a method of loading liposomes with activesubstances, wherein the liposomes are made permeable at a well-definedpH value and sealed.

The invention also relates to the use of the liposomes in the productionof nanocapsules.

The invention also relates to the use of the liposomes in the productionof release systems in diagnostics.

Advantageously, the liposomes are used for the transport and/or releaseof active substances.

In another embodiment, the liposomes conveniently are used as depotformulation and/or as circulative depot.

Advantageously, the liposomes can be used in intravenous or peritonealapplication.

In another embodiment of the invention, the liposomes are used withadvantage as vector to transfect cells in vivo, in vitro and ex vivo.

Surprisingly, it has been determined that the permeability of the lipidlayer of the inventive liposomes particularly depends on the pH valueand thus, on the state of charge of the sterol derivative. In addition,when using the well-known CHEMS, an increase in permeability occurs onlyin the simultaneous presence of high amounts of phosphatidylethanolamine (PE) in the membrane. This phospholipid does not formmembranes by itself, being stabilized artificially by CHEMS. However,one drawback of such liposomes is their low stability which can be seenin the fact that smaller molecules of active substance slowly diffuseout even without a change in pH.

When using His-Chol, in particular, the membranes comprised ofphosphatidyl choline (PC) are made permeable in such a way thatentrapped active substances or markers will diffuse out within minutesto hours. However, these membranes themselves are stable, showing lowinitial permeability. Liposomes using the structures according to theinvention are therefore suited to construct release systems whereinrelease of active substances is to proceed in dependence on the pH valueof the medium.

Surprisingly, it has also been found that amounts of proteins or DNAabove average can be enclosed in liposomes including the compoundsdescribed herein in the membranes thereof. The efficiency of suchincorporation depends on the pH value of the solution employed.Therefore, a process for efficient encapsulation of proteins or DNA inliposomes can be performed by initially adjusting a pH value that wouldresult in good binding of the cargo molecules to the liposomes. With DNAas polyanion, low pH values of about 4 to 5 are used. With proteins, auseful pH value will depend on the isoelectric point of the protein,which should be below the pKa value of the substance according to theinvention. Encapsulation is particularly effective when the pH value ofthe medium is selected so as to range between the isoelectric point ofthe protein and the pKa value of the sterol derivative. The proteinsthen will have a negative charge, while the lipid layer already has apositive net charge. If necessary, non-incorporated cargo moleculesadhering on the outside can be removed by simply increasing the pHvalue. This step is necessary in all those cases where non-incorporatedcargo molecules would give rise to aggregation of the liposomes. Oneadvantageous fact when using the components of the invention is that theentrapped active substances must be maintained under conditions allowinginteraction with the lipid layer only during the period of actualenclosure. Once the lipid layer remains closed in itself, it is possibleto change to other conditions. Thereby, possible inactivation of activesubstances, particularly of proteins, can be minimized.

Liposomes comprising the components of the invention can be coated withpolymers under conditions well-known to those skilled in the art, wheresingle or multiple deposition of such substances on the surface ispossible, in particular. In multiple deposition, optionally in thepresence of crosslinkers, liposomal nanocapsules are formed as describedin WO 00/28972 or WO 01/64330.

One advantageous fact when using the substances described herein is thatthe electrostatic interaction with the polyelectrolyte can beinterrupted. As is well-known, the interaction of a polyelectrolyte withcharge carriers of the liposomal membrane may give rise to demixing ofmembrane components and formation of lipid clusters. In many cases, suchdemixing is accompanied by a permeabilization of the liposomes. Thesubstances of the invention allow for elimination of this interactionfollowing the coating process. When increasing the pH value at thispoint, the liposomes will be entrapped in the nanocapsules merely in asteric fashion, and interaction between the membrane andpolyelectrolytes does no longer exist. In this way, cluster formation oflipids and associated permeabilization of the membrane can becircumvented.

In one variant of the teaching according to the invention, these changesin permeability are used in a well-directed fashion in loadingliposomes. To this end, an active substance to be enclosed can be addedto a medium under conditions of high permeability, followed by adjustingconditions of low permeability. In this way, the active substance willremain inside the liposomes. Thereafter, non-entrapped active substancecan be removed, if necessary. Such changes in permeability can beinduced on liposomes or on liposomal nanocapsules.

Surprisingly, it has also been found that liposomes including e.g.His-Chol or Pip-Chol in the membranes thereof are capable of chelatingmetal ions. This property results in an increase of the positive chargeof the liposome. This effect is observed to be particularly strong atneutral pH values, because the inherent charge of the compound is low inthis case. Owing to their chelating properties, such liposomes can beused in biochemical diagnostics and in pharmaceutical therapy.

In a detection system, such liposomes can be loaded with metal ionswhose fluorescence is enhanced by chelate formation, i.e., terbium oreuropium ions, for example. Liposomes for such uses additionally includecomponents determining the specificity, i.e., antibodies, lectins,selectins, receptors, or hormones, or RNA aptamers. In a particularlypreferred embodiment of the use according to the invention, the presenceof these metal ions is restricted to the volume of the liposomes so asto avoid non-specific signals from slowly released metal ions adheringon the outside. Coupling of pyridyldithioethenylamine to CHEMS providesa compound with a special combination of desirable properties. Theterminal pyridyl group results in significant positive charging of themembrane even under mild conditions (pH 6 to 7). Liposomes producedusing this compound are capable of binding proteins or nucleic acids inlarge amounts above average. By reducing the disulfide bond, e.g. usingdithiothreitol or tris(2-carboxyethyl)phosphine, a free thiol functionis generated, resulting in neutralization of the surface. Under theseconditions, such enhanced binding of proteins or nucleic acids isdecreased. Ultimately, it is completely lost when increasing the pHvalue, because the formation of thiolate ions results in a negativelycharged surface.

In those cases where biological macromolecules possess thiol functionsof their own, binding thereof can be retained. This is the case withnumerous proteins. Where other materials are concerned, a person skilledin the art will be familiar with procedures of introducing free thiolfunctions in such molecules, while retaining the biological activitythereof (G. Hermanson, Bioconjugate Techniques). Substances including afree thiol function can be fixed covalently to the surface of such lipidlayers by means of a disulfide exchange reaction.

Owing to their particularly favorable properties in binding and couplingof proteins, liposomes including PDEA-Chol are especially suitable inthe production of nanocapsules on liposomal templates such as describedin WO 00/28972.

Surprisingly, it has also been found that liposomes including PDEA-Cholare capable of changing their permeability in accordance with the redoxstate. Reductive removal of the pyridyl group results inpermeabilization of the membrane.

Surprisingly, it has also been found that the liposomes according to theinvention readily undergo fusion with other membranes at low pH values.In general, this step requires the presence of a larger amount of PE inthe membrane. As a result of its tendency of forming hexagonal phases,said PE assumes the function of a helper lipid. However, the inferiorstability of such membranes is disadvantageous, and gradual release ofentrapped active substances is frequently observed.

However, liposomes produced using the substances according to theinvention undergo effective fusion even in the absence of such a helperlipid. Thus, when using the substances of the invention, it is possibleto produce liposomes which are capable of stably encapsulating an activesubstance, but undergo fusion with cell membranes under the conditionsof low pH values to release the active substance there.

This combination of two properties is an important precondition for theincorporation of cargo molecules in cells. In fusion of liposomes withcell envelopes or components, the aqueous volumes of both partnerscombine, with no opening of the membrane structures to the medium takingplace. As a result, uncontrolled influx or efflux of other substances isavoided.

One essential precondition for the use of liposomes for experimental ortherapeutic purposes is their compatibility with cells and tissues. Anumber of well-known compounds used to incorporate DNA or proteins incells (for example, the cationic lipid DOTAP) are cytotoxic.

Surprisingly, it has also been found that the compounds of the inventionexhibit reduced cytotoxicity. These measurements will be illustrated inthe experimental section. Thus, compared to the commonly used DOTAP,His-Chol shows lesser toxic effects in the MTT test.

Another precondition for the construction of vectors to be used in geneor protein transport into cells is their compatibility with serum orblood. Due to their strong cationic charge, vectors known at presentform uncontrollable large aggregates, resulting in formation of thrombiin the organism. Their use in vivo is therefore practically impossibleand is restricted to in vitro or ex vivo applications.

Surprisingly, it has been found that liposomes constructed using thecomponents of the invention do not form any aggregates in serum orblood.

Another precondition for the construction of vectors to be used inprotein or gene transfer is their stability under physiologicalconditions. Upon application into the blood circulation, liposomes areattacked by components of the complement system and undergo rapid lysis.This reaction proceeds within minutes. As a result, pores are formed inthe membrane, which allow even large molecules such as proteins todiffuse out therethrough. At present, stabilization of liposomes withrespect to this mechanism is only possible by incorporating cholesterolin the lipid layer. While such liposomes are highly stable, they are nolonger able to interact with cells or readily release their activesubstance. Surprisingly, it has been found that liposomes constructedusing the components of the invention are stable in serum or blood forseveral hours. Even under such conditions, the release of activesubstance is low.

A liposomal vector for the transport of active substances must satisfyat least three preconditions: it must have low toxicity, entrap theactive substance firmly and stably, and be compatible with serum orblood.

All of these three preconditions are satisfied by liposomes producedusing the sub-stances according to the invention. The liposomesdisclosed herein are therefore well suited for therapeutic uses. Otherproperties supporting such uses are good loadability with activesub-stances and well-directed release of these substances bypermeabilization of the membrane at suitable pH values or redox states.

Without intending to be limiting, the invention will be explained inmore detail with reference to the following examples.

EXAMPLE 1 Synthesis of His-Chol

1.31 g of cholesterol hemisuccinate is dissolved in 20 ml of DMF at roomtemperature. The solution is added with 438 mg of carbonyldiimidazoledissolved in 20 ml of DMF. The mixture is allowed to stir for 1 hour andsubsequently added with 300 mg of histamine. The mixture is stirredovernight and concentrated thoroughly in vacuum. The residue is purifiedby column chromatography on silica (Kieselgel 60), withchloroform/methanol 10:1 being used as eluant. (Yield 54%; 1.45 mmol),pure in HPLC, identity determined using MS and ¹³C-NMR.

EXAMPLE 2 Synthesis of PDEA-Chol

The procedure for the synthesis of PDEA-Chol is as above. Instead ofhistamine, 600 mg of pyridyldithioethaneamine hydrochloride is used.

EXAMPLE 3 Synthesis of Mo-Chol

The procedure for the synthesis of Mo-Chol is as above. Instead ofhistamine, 350 mg of 4-(2-aminoethyl)morpholine is used.

EXAMPLE 4 Preparation of Cationic pH-Sensitive Liposomes

5 mg of His-Chol and 9.8 mg of POPC are dissolved in 4 ml ofchloroform/methanol (1:1 v/v) and dried completely in a rotaryevaporator. The lipid film is hydrated with 4.3 ml of a correspondingbuffer (10 mM Kac, 10 mM HEPES, 150 mM NaCl, pH 7.5) at a lipidconcentration of 5 mM using brief ultrasonic treatment (5 minutes).Finally, the suspension is frozen and, following thawing, subjected tomultiple extrusions (Avestine LiposoFast, polycarbonate filter, porewidth 200 nm).

The profile of the zeta potential at various pH values is illustrated inthe Table below.

pH value Zeta potential in mV 4.4 +52 6.2 −3 7.5 −13

EXAMPLE 5 Permeability

Liposomes are produced generally as in Example 4. The following lipidmixtures are used (figures in mole-%)

A: DPPC 60 His-Chol 40 B: DPPC 60 CHEMS 40 C: POPC 60 His-Chol 40 D:POPC 60 CHEMS 40

The lipids are dissolved in the solvent mixture as indicated and driedunder vacuum. The lipid films are hydrated with 100 mMcarboxyfluoresceine, 50 mM NaCl, pH 7.5, at a lipid concentration of 15mM and frozen, thawed and extruded as above. Non-entrappedcarboxyfluoresceine is removed by gel filtration.

20 μl of the liposomes thus obtained are incubated with 2 ml of buffer(10 mM potassium acetate, 10 mM HEPES). After 90 min, the amount ofdischarged carboxyfluoresceine is determined by measuring thefluorescence intensity of the sample. A comparative sample with completerelease of the entrapped marker is obtained by addition of 0.2% TritonX-100 to the batch.

A B C D pH 4.4 8 38 80 39 pH 5.4 7 7 35 18 pH 6.4 6 9 18 16 pH 7.4 7 717 15 pH 8.4 5 11 15 15

EXAMPLE 6 Chelating of Metal Ions

Liposomes are produced as in Example 4. 40 μl of these liposomes aresuspended in 7 ml of buffer (10 mM potassium acetate, 10 mM HEPES, pH4.2 or pH 7.5). Subsequently, the metal ions are added with theconcentrations as indicated, and the zeta potential of the liposomes ismeasured.

Ions pH 4.2 pH 7.5 Ni²⁺ 10 mM +16.8 11.6 Ca²⁺ 10 mM +40.5 +4.6 Zn²⁺ 10mM +65.4 not measurable No addition +47.6 +2.4

At neutral pH, chelating of nickel ions is clearly detectable. Atslightly acidic pH, nickel ions are not bound anymore, but zinc ionsare. The non-transition metal calcium behaves indifferently, forming nochelate complexes.

EXAMPLE 7 Binding of DNA

1 mg of DNA (herring sperm, SIGMA D3159) is dissolved in 1 ml of water.Using the liposomes from Example 4, a 0.2 mM suspension in buffer (10 mMpotassium acetate, 10 mM HEPES, pH 4.2 or pH 7.5) is produced. 45 μl ofDNA solution each time is added to 1 ml of these different liposomesamples and mixed rapidly. After 15 minutes of incubation, the sample isfilled up with 6 ml of the corresponding buffer, and the zeta potentialof the liposomes is measured.

Zeta potential in mV No DNA DNA added pH = 4.2 +27.1 −45.7 pH = 7.5 −6.3−39.6

EXAMPLE 8 Fusion Properties

Liposomes having the following compositions are produced as in Example 1(all figures in mole-%)

A) POPC 60 His-Chol 40

X) POPC 100

Y) POPC 60 DPPG 40

The optionally cationic liposomes A are incubated with the neutralliposomes X or with the anionic liposomes Y in buffer (10 mM HEPES, 10mM potassium acetate, pH 4.2 or 7.5). Possible fusion of liposomes isanalyzed using size measurement by means of dynamic light scattering.

Liposome 1 X Y Liposome 2 A A pH 4.2 181.6 nm 1689.3 nm pH 7.5 191.8 nm 250.0 nm

The initial values of the liposomes were 161.8 nm at pH 4.2 and 165.9 nmat pH 7.5

X) 199.2 nm

Y) 183.2 nm

The size of the YA pair of complementary charge is clearly differentfrom the size of the mixed suspensions including the neutral liposomeXA. The degree of interaction is determined by the charge level of theoptionally cationic liposomes. Fusion to form larger units does notdepend on the fusogenic PE lipid.

EXAMPLE 9 Permeability to Macromolecules

15 μmol of DOPE and 10 μmol of His-Chol are dissolved in isopropanol,and the solvent is removed under vacuum. The dried lipid film is addedwith 2.5 ml of a solution of proteinase K in buffer (1 mg/ml proteinaseK, 10 mM potassium acetate, 10 mM HEPES, 150 mM NaCl, pH 4.2). Followinghydration of the film, the liposomes having formed are extruded througha 400 nm membrane. Non-entrapped proteinase is removed by flotation ofthe liposomes in a sucrose gradient.

The liposomes thus produced are incubated with 7.5 ml of buffer at pH4.2 and pH 7.2 (buffer as above, initial pH 4.2 and 8.0). Followingincubation, the liberated proteinase K is separated by ultrafiltrationusing a 0.1 μm membrane. The liposomes remaining in the filter are thentreated with 7.5 ml of a solution of Triton X-100 in buffer (as above,pH 8.0).

All of the filtrates are tested for presence of proteinase K. To thisend, a solution of azocasein (6 mg/ml azocasein in 1 M urea, 200 mM Trissulfate, pH 8.5) is used. 500 μl of this solution is mixed with 100 μlof filtrate or buffer and incubated for 30 minutes at 37° C. Thereaction is terminated by addition of 10% trichloroacetic acid.Precipitated proteins are removed by centrifugation. The coloration inthe supernatant is measured at 390 nm.

Absorption at 390 nm - pH Incubation Triton X-100 blank 4.2 − 0.01654.2 + 0.1731 7.2 − 0.1354 7.2 + 0.0260

When incubating the liposomes at a pH value of 4.2, no or only a smallamount of proteinase K is liberated. The enzyme is liberated only afterdissolving the liposomes with Triton X-100.

When incubating the liposomes at a pH value of 7.2, a major amount ofthe enzyme is liberated even without addition of Triton and will befound in the first filtrate. Addition of Triton then is barely capableof leaching further enzyme from the liposomes.

EXAMPLE 10 Cytotoxicity

The toxic effect of the substances was investigated using the MTT test.To this end, HeLa cells were seeded at a density of 2×10⁴ cells percavity of a 96-well titer plate and cultured for two days. Liposomes ofvarying composition (see Table) were added to the cells at aconcentration of 0.5 mM and incubated with these cells for 24 hours.Subsequently, the MTT test was performed.

Liposome Viability (MTT) Notes — 100%  DOPE 60/DOTAP 40 82% DOPE60/DC-Chol 40 57% Cells detaching DOPE 60/His-Chol 40 92% DOPE60/Mo-Chol 40 103%  POPC 60/His-Chol 40 90%

DOPE: Dioleoylphosphatidyl ethanolamine, 60 mole-% each time POPC:Palmitoyloleoylphosphatidyl choline, 60 mole-% each time

DC-Chol: N,N-Dimethyl(2-aminoethyl)carbamoylcholesterol

Even at high concentrations, the liposomes comprising the substances ofthe invention are well-tolerated by cells. Toxic effects are barelydetectable. In contrast, well-known cationic lipids such as DC-Chol orDOTAP have a significant cytotoxic effect.

EXAMPLE 11 Stability in Serum

Carboxyfluoresceine-loaded liposomes having the compositionsPOPC/His-Chol 60:40, POPC/Mo-Chol 60:40, and POPC/DPPG 60:40 (allfigures in mole-%) were produced in analogy to Example 5. Formeasurement, the liposomes were diluted to 0.1 mM in human serum andincubated at 37° C. Fluorescence was measured at specific intervals.Complete liberation was achieved by addition of Triton X-100 to themeasuring buffer. The CF liberation data are summarized in the Tablebelow. For comparison, the negatively charged POPC/DPPG liposomesvirtually losing no CF during 4 hours are illustrated. POPC/His-Cholliposomes show high serum stability up to 2 hours, but lose some CFafter 4 hours. POPC/Mo-Chol liposomes exhibit a somewhat higherpermeability than POPC/His-Chol.

POPC/His-Chol POPC/Mo-Chol POPC/DPPG 0 min 0% 0% 0% 30 min 0% 4% 0% 60min 2% 5% 1% 120 min 4% 9% 2% 240 min 19%  16%  2%

EXAMPLE 12 Transfection into Cells

HeLa cells (3×10⁵) were placed in each cavity of a 6-well titer plateand cultured for three days. Liposomes having the same compositions asin Example 10 were produced in the presence of fluorescence-labelleddextran (TRITC dextran, 10 mg/ml in hydration buffer). Non-incorporatedTRITC dextran was removed by gel filtration. The liposomes thus producedwere added to the cells and incubated for 6 hours at 37° C.Subsequently, the cells were washed twice with buffer. Dextran uptakewas monitored by microscopic imaging and quantified using fluorescencespectroscopy.

The results are summarized in the following Table and in FIG. 2.

TRITC dextran uptake Liposome in μg - (TRITC dextran in buffer) 0.1 DOPE60/DOTAP 40 1.5 DOPE 60/DC-Chol 40 1.1 DOPE 60/His-Chol 40 0.3 DOPE60/Mo-Chol 40 0.7 POPC 60/His-Chol 40 0.4

The new compounds do not quite achieve the efficiency of the well-knowncationic lipids DOTAP or DC-Chol. However, they are also capable ofmediating the transfection of macromolecules into cells. It is to beexpected that the efficiency can be increased substantially when usingligands for cell adhesion.

1. A sterol derivative according to general formula (I):cation-spacer 2-Y-spacer 1-X-sterol (1), wherein: said cation is anitrogen base selected from the group consisting of piperazines,imidazoles, morpholines, purines, pyrimidines, and pyridines; saidspacers 1 and 2 are independently linear, branched or cyclic C₁₋₈ alkyl,and comprise 0-2 ethylenically unsaturated bonds, wherein at least oneof said spacers 1 and 2 is cyclic C₁₋₈ alkyl; said linking group X isselected from the group consisting of —(C=0)-O— and —(C=0)-NH—; saidlinking group Y is selected from the group consisting of —O-(0=C)—,—NH-(0=C)-1-(C=0)-O—, and —(C=0)-NH—; said sterol is selected from thegroup consisting of cholesterol, sitosterol, campesterol, desmosterol,fucosterol, 22-ketosterol, 20-hydroxysterol, stigmasterol,22-hydroxycholesterol, 25-hydroxycholesterol, lanosterol,7-dehydrocholesterol, dihydrocholesterol, 19-hydroxycholesterol,5α-cholest-7-en-3β-ol, 7-hydroxycholesterol, epicholesterol, ergosterol,and dehydroergosterol; and said sterol derivative has a pKa value ofbetween about 3.5 and about
 8. 2. The sterol derivative according toclaim 1, wherein at least on of said spacers 1 and 2 have hydroxylgroups.
 3. The sterol derivative according to claim 1, wherein thesterol derivative has a pKa value of between about 4 and about
 7. 4. Aliposome comprising the sterol derivative of claim
 1. 5. The liposome ofclaim 4, wherein said liposome comprises between about 5 mole-% andabout 50 mole-% of sterol derivatives.
 6. The liposome of claim 5,wherein said liposome comprises between about 5 mole-% and about 40mole-% of sterol derivatives.
 7. The liposome of claim 6, wherein saidliposome comprises between about 10 mole-% and about 30 mole-% of sterolderivatives.
 8. The liposome of claim 4, wherein the liposome comprisesone or more lipids selected from the group consisting of phosphatidylcholine, phosphatidyl ethanolamine, and diacylglycerol.
 9. The liposomeof claim 8, wherein said liposome is neutral or negatively charged at apH of from about 7.0 to about 7.8.
 10. The liposome of claim 4, whereinsaid liposome has an average size of between about 50 and 1000 nm n. 11.The liposome of claim 10, wherein said liposome has an average size ofbetween about 50 and 300 nm.
 12. The liposome of claim 11, wherein saidliposome has an average size of between about 60 and 130 nm.
 13. Theliposome of claim 4, wherein said liposome further comprises an activesubstance.
 14. The liposome claim 13, wherein said active substance isselected from the group consisting of a protein, a peptide, a DNA, anRNA, an antisense nucleotide, a decoy nucleotide, and a mixture thereof.15. The liposome of claim 13, wherein at least about 80% of said activesubstance is situated inside the liposome.
 16. A method of loading theliposome of claim 13 with an active substance, said method comprising:encapsulating said active substance in said liposome at a binding pHvalue; and removing unbound active substances at a second pH value. 17.A method of loading the liposome of claim 13 with an active substance,said method comprising: permeabilizing said liposome by treatment at apH value sufficient to enable loading of said active substance, andsealing said liposome.
 18. A method for the transport and release of anactive substance in a subject, said method comprising administering tosaid subject the liposome of claim
 13. 19. The method of claim 18,wherein said administration is intravenous or peritoneal.
 20. Atransport and release system for the transport and release of an activesubstance in a subject, said system comprising the liposome of claim 13.21. A depot formulation or circulative depot comprising the liposome ofclaim
 13. 22. A nanocapsule prepared from the liposome of claim
 4. 23. Avector for transfecting cells in vivo, in vitro or ex vivo, said vectorcomprising the liposome of claim 4 and a nucleic acid.